42-3 |
Development of bio-ligand for detection of meat and bone meal using phage display |
S. H. KIM1, M. A. Vanchina1, V. A. Petrenko2, C. I. Wei3, and H. An4. (1) Dept. of Nutrition & Food Science, Auburn Univ., 328 Spidle Hall, Auburn, AL 36849-5605, (2) Dept. of Pathobiology, Auburn Univ., 166 Greene Hall, Auburn, AL 36849, (3) Dept. of Nutritional Sciences, Oklahoma State Univ., 139 Human Environmental Sciences Bldg., Stillwater, OK 74078-6141, (4) School of Pharmacy, Univ. of Southern California, 1985 Zonal Ave., B-4 Pharmaceutical Sciences Ctr., Los Angeles, CA 90089-9121 Meat and bone meal (MBM) is widely utilized to supplement nitrogen in animal feed. Unfortunately, MBM contaminated with a transmissible spongiform encephalopathy (TSE) agent has been known as the common source of bovine spongiform encephalopathy transmission. Thus, ruminant derived protein in animal feed has been banned. Numerous efforts have been exerted to develop a method to detect MBM contaminated in animal feed. The difficulties have been encountered because of the extensive degree of hydrolysis of the rendered proteins in MBM resulting from high temperature/high pressure treatment. In this study, we approached phage display technique, since peptide display on the surface of a filamentous bacteriophage has brought to attention as a simple and powerful approach to identify a variety of biomolecules, such as enzymes, carbohydrate moieties, and receptors. The objective of this study was to develop bio-ligand using phage display technique for detection of MBM in animal feed. M13 phage display peptide library was subjected to screening with MBM and bovine smooth muscle extract. Specifically bound phages were eluted, infected into Escherichia coli, and amplified. After three rounds of consecutive selections, individual phage clones were isolated and sequenced to identify the peptide responsible for binding. Binding affinity of the peptides to bovine smooth muscle and MBM extracts was screened using the ELISA assay. After the affinity selection, the phage clones showing the high binding affinity to MBM extract were obtained. The consensus sequences of displayed peptide were Ser-Met-Lys-Thr-Asn-Gln-Pro and Ser-Met-Lys-Thr-Asn-Ala-Ala. The clones showed the detectable ELISA signal (>O.D. 0.5) at 109 pfu/ml of phage. Phage display was an effective method to develop bio-ligands to react with MBM. The selected clones showed the potential to detect MBM in animal feed.
Session 42, Food Chemistry: Proteins II
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